Mammalian melanocytes can produce two types of melanin, eumelanin (which is black and/or brown in color) and pheomelanin (which is red and/or yellow in color) (Prota, 1992; Ito, 1993a). Switching between these two types of melanins in follicular (i.e. hair bulb) melanocytes elicits a temporary shift from eu- to pheomelanogenesis, which is responsible for the wild-type agouti pigment pattern of murine hair color, i.e. a yellow striped band against a black background on each hair shaft, as reviewed by Silvers (1979) and Hirobe (1991). This physiological switch is controlled by the agouti locus, which has recently been cloned (Bultman et al., 1992; Miller et al., 1993). The agouti locus-encoded protein is thought to be secreted by nonmelanocytic follicular cells (Silvers, 1958) and functions as an antagonist of the melanocyte-stimulating hormone (xcex1MSH) receptor, which is expressed specifically by melanocytes (Lu et al., 1994). The recessive black mutation (nonagouti; a) at the agouti locus results in a nearly complete loss of agouti RNA which causes the constitutive production of eumelanin black hairs. In contrast, the dominant lethal yellow mutation (Ay/a) elicits the ubiquitous ectopic production of agouti RNA in nearly all tissues of the body and throughout the entire phase of the hair growth cycle, prompting the production of completely yellow pheomelanic hairs (Ito and Fujita, 1985; Duhl et al., 1994a,b).
For many decades, melanosomal proteins that regulate melanin biosynthesis have been studied and characterized, especially those required for eumelanogenesis, as reviewed by Hearing and Tsukamoto (1991) and Hearing and King (1993). Tyrosine (EC 1.14.18.1), which is encoded at the albino locus, is the essential enzymatic protein for both types of melanin formation. Tyrosinase is a trifunctional enzyme with three catalytic activities: tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA), the oxidation of DOPA to dopaquinone and the oxidation of 5,6-dihydroxlindole (DHI) to indole-5,6-quinone (Korner and Pawelek, 1982; Hearing, 1987; Tripathi et al., 1992). Other tyrosine-related proteins (TRP) have been shown to regulate eumelanogenesis catalytically at steps distal to tyrosinase. TRP1, encoded at the brown locus, functions as 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase (Jimenez-Cervantes et al., 1994; Kobayashi et al., 1994b) while TRP2, encoded at the slaty locus, functions as DOPAchrome tautomerase (EC5.3.2.3) (Barber et al., 1984; Aroca et al., 1990; Tsukamoto et al., 1992; Jackson et al., 1992). The silver locus-encoded protein had been proposed to function in melanogenesis catalytically within the melanosome, and although it has some limited homology to the tyrosinase-related proteins (Kwon et al., 1991), it has been recently demonstrated to be a melanosomal matrix protein and to have none of the known melanogenic activities (Zhou et al., 1994; Kobayashi et al., 1994a). The product of the pink-eyed dilution locus is also a melanosomal protein that actively participates in the regulation of melanogenesis (Tamate et al., 1989; Chiu et al., 1993; Rosemblat et al., 1994).
During pheomelanogenesis, the activity and expression of tyrosinase has been reported to be lower than that found during eumelanogenesis (Barber et al., 1985; Burchill et al, 1986, 1989; Lamoreux et al, 1986; Movaghar and Hunt, 1987; Tamate et al., 1989; Granholm et al., 1990; Kappenman et al., 1992). In addition to tyrosinase, thiols are essential to capture the dopaquinone made enzymatically by tyrosinase in order to produce the cysteinyldopas necessary for pheomelanogenesis (FIG. 1). Subsequent cyclization and polymerization of cysteinyldopas in an uncharacterized series of reactions results in the production of the high molecular mass complex known as pheomelanin (Prota, 1992; Hearing and King, 1993; Ito, 1993 a). The switch between eu- and pheomelanogenesis has been proposed to be regulated enzymatically primarily at the level of tyrosinase (Ito, 1993a). The potential roles of other melanogenic gene products during pheomelanogenesis, however, remain unclear, since there have been few studies about the expression and function of such proteins during pheomelanogenesis. To date, only the absence of DOPAchrome tautomerase activity in yellow mice has been reported (Barber et al., 1985), as has the absence of TRP1 mRNA expression in pheomelanogenic mice (Thody and Burchill, 1992) and human melanoma cells (Del-Marmol et al, 1993).
The present invention determines the transcriptional and translational levels of the expression and catalytic functions of tyrosinase, TRP1, TRP2 and the silver protein during pheomelanogenesis. The expression and melanogenic activities of those proteins in hair bulbs of wild type agouti mice during their pheomelanogenic phase is also determined in the present invention. The present invention shows that TRP1, TRP2 and the silver protein function specifically in eumelanogenesis and may play an important role in the production of eumelanosomes. The down-regulation of expression of those proteins during melanogenesis is shown in the present invention using agouti signaling protein.
The present invention is a biologically active peptide of the Agouti Signaling Protein which has depigmenting activity.
The present invention is a method of down-regulating one or more melanogenic enzymes involved in melanin synthesis.
The present invention is also the use of the Agouti signaling protein and biologically active peptides thereof in methods of inhibiting melanin synthesis.
A further aspect of the invention is the treatment of hyperpigmentary conditions and diseases using an effective amount of agouti signaling protein or peptides thereof.
Another aspect of the invention is a pharmaceutical composition of agouti signaling protein or biologically active peptides thereof and a pharmaceutically acceptable carrier.
The present invention is also a method for screening for biologically active peptides of the agouti signaling protein and other compounds useful in inhibiting melanin synthesis.